Most investigators have assumed that receptors for GnRH are located on the plasma membrane of gonadotrophs. However, recent studies using the immunoperoxidase staining technique and density gradient centrifugation indicate that receptors for GnRH are also associated with membranes of secretory granules in gonadotrophs. The proposed research is designed to determine the subcellular distribution of receptors for GnRH in homogenates of ovine adenohypophyseal tissue. Following purification of each subcellular fractions to specifically bind radioiodinated GnRH will be determined. In addition, we will attempt to determine if GnRH is initially associated with receptors on the plasma membrane and later translocated to the interior of the cell. These studies will be conducted utilizing high voltage electron microscopic autoradiography to observe the distribution of silver grains over various subcellular organelles of intact gonadotrophs that have been incubated with radioiodinated GnRH in vivo and in vitro for various lengths of time. The second phase of the proposed research is designed to determine if estradiol-17 beta and progesterone influence the sensitivity of the pituitary to GnRH by controlling the synthesis or affinity of the receptors for this decapeptide. For these studies, binding of radioiodinated GnRH to anterior pituitary glands of normally cycling ewes or anestrous ewes pre-treated with estradiol-17 beta and/or progesterone will be quantified. The data will be analyzed by Scatchard plot to determine changes in numbers or affinity of receptors for GnRH. The final phase of the research will be directed toward elucidating the mechanisms whereby analogs of GnRH exert their stimulatory or inhibitory properties. In this respect we propose to correlate the affinities with which various analogs are bound to the GnRH receptor with the respective abilities of the analogs to inhibit the GnRH-induced release of LH (inhibitory analog) or to release LH (stimulatory analog) from slices of adenohypophyseal tissue incubated in vitro. It is hoped that the proposed research will lead to information which may be used to develop more efficient, and safer, methods of regulating fertility in humans, food producing animals and pets.